Slime control in industrial waters

ABSTRACT

Slime formation is controlled (e.g., retarded or removed) by the intentional addition to industrial waters (e.g., white water in pulp and paper mills) of slime controlling amounts of the enzyme levan hydrolase.

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0 132 United Sta [111 3,773,623 Hatcher et al. 1 Nov. 20, 1973 SLIMECONTROL IN INDUSTRIAL [56] References Cited WATERS UNITED STATES PATENTS[75] Inventors: Herbert J. Hatcher, Bloomington; 3,654,086 4/1972 Ziffer195/63 1 al I Robert J. Truda, St. Paul; Thomas G. Lechner, Lake Elmo;Charles R. McDuff, St. Paul, all of'Minn.

[73] Assignee: Economics Laboratory, Inc., St.

Paul, Minn.

[22] Filed: Feb. 25, 1972 21 Appl. No.: 229,596

Related US. Application Data [62] Division of Ser. No. 69,933, Sept 4,1970.

OTHER PUBLICATIONS Avigad et al., Meth in Eng Vol. Vlll, No. I06 FructanHydrolasis," p.621-628, 1966. QP6OIC72 Primary Examiner-Lionel M.Shapiro Att0rneyMerchant & Gould Y [57] ABSTRACT Slime formation iscontrolled (e.g., retarded or removed) by the intentional addition toindustrial waters (e.g., white water in pulp and paper mills) of slimecontrolling amounts of the enzyme levan hydrolase.

3 Claims, No Drawings [52] US. Cl. 195/60, 106/15 AF, 162/190,210/1,162/161 [51] Int. Cl B321) 27/40 [58] Field Of Search 195/31, 62,64, 65, 195/66, 68, 57, 52, 60; 162/161, 190; 99/96; v 106/15 AF SLIMECONTROL IN INDUSTRIAL WATERS CROSS-REFERENCE TO RELATED APPLICATIONSThis is a division of application Ser. No. 69,933, filed Sept. 4, 1970entitled SLIME CONTROL IN INDUS- TRIAL WATERS BACKGROUND OF THEDISCLOSURE The formation of slime in industrial waters is a majorindustrial problem.

The term slime is a broad one covering a wide range of viscous, mucous,or leathery materials and mixtures found in industrial waters. Slimesare frequently polymeric in nature and can be broadly classified aschemical, biological, or composite slimes depending upon their cause orcomposition. For example, raw materials and equipment used in the paperindustry are not sterile and water used in conjunction with suchequipment is continuously being contaminated with a wide variety ofmicroorganisms from such sources as wood pulp, chemicals, air, make-upwater, and the like. The existing conditions of temperature and the likepermit the growth of a wide range of microorganisms. The growth ofcertain specific forms of these biological contaminants causes orproduces polymeric excretions or products that are or become slime.

Historically, slime formation has been controlled by the addition toindustrial waters (e.g., white water associated with the pulp and paperindustry) of slimicides. The purpose of these slimicides is to destroyor arrest the growth of some of the many organisms present in the waterto thereby prevent or retard the formation of slime. Slimicides (ortoxicants as they are sometimes called) fall into two generalcategories; the first, biocides; and the second, biostats. Chemicalsused as slimicides have included chlorine, phenylmercuric acetate,pentachlorophenol, tributyl tin oxide, and isothiocyanates, all of whichare relatively toxic to humans.

Many workers have investigated the causes of slime formation inindustrial waters and it is generally recognized that a broad spectrumof bacteria, predominately gram-negative bacteria, are responsible forthe formation of slimes, particularly paper mill slimes. Yeasts andmolds are lesser causes of slime than bacteria, particularly in papermills. For example, although molds are easily isolated from industrialwaters and cultivated in the laboratory, rapid natural fungal growthfrom mixed cultures in recirculating industrial waters has not beenobserved.

Those engaged in the search for slimicides or toxicants haveperiodically observed the lack of correlation between the totalbacterial counts and slime accumulation. Frequently, high bacterialtotal counts have been noted in environments in which no significantslime build-up have been observed. Similarly, low bacterial total countshave been observed in conjunction with substantial accumulations ofslime. These observations have been a source of concern to those engagedin the water treatment industry.

SUMMARY OF THE INVENTION We have observed that levan (a specific type offructose polymer which is produced by a wide variety of bacteria) is asignificant component of many industrial slimes. We have furtherdiscovered that it is difficult and economically impractical to attemptto destroy or arrest the growth of all common bacteria that producelevan. We have found that slime accumulation can be controlled morereadily by the addition of the enzyme levan hydrolase to the industrialwater to thereby hydrolyze the levan produced by the bacteria. Althoughthe enzyme may be used in conjunction with biocides and biostats,effective control over slime formation can be obtained by the mere useof this specific enzyme (i.e., levan hydrolase).

DETAILED DISCUSSION Industrial Waters and the Slime Problem Industrialwaters comprise those waters used in industrial plants for such purposesas conveying particulate material (e.g., paper pulp), cooling, and thelike. Although the present invention is applicable to a wide range ofindustrial waters in which slime formation characteristically occurs,the present invention is particularly useful in the treatment of whitewater associated with pulp and paper mills. Consequently, the presentinvention will be described with particular reference to the treatmentof slime formations in paper mill water systems without intending to belimited thereby.

Various persons have reviewed the slime problem in the paper industrycaused by microorganisms present in white water. Attention is directedto the following articles and the references cited therein:

Coster, E., The Slime Problem in the Paper Industry Caused byMicroorganisms, Appita 2l No. 4: 131-138 (January, 1968).

Leckey, C. R., The Slime Board Method of Paper Mill Toxicant Evaluation,Tappi 43, No. 9: 781-783 (September, 1960).

Michalski, R. J. et al, A Method for Determining The Effect ofDispersants in Slime Control Performance, Tappi 46, No. 2: 167a-l72a(February, 1963).

We have observed, as have prior workers, that the degree of slimeformation cannot be directly correlated with total microbial countsprobably because white water contains a broad spectrum ofmicroorganisms. However, the presence ofa broad spectrum ofmicroorganisms in such industrial waters is responsible for theactivities of other workers in attempting to identify or producechemicals having broad biocidical or biostatic properties.

In the course of our work, we found that a common type of polymerproduced by many species of bacteria is a polysaccharide known as alevan. A levan is a polymer made up of fructose units joined by beta 2,6linkages. We decided to approach the problem of slime accumulation by adirect attack on the slime, particularly upon levan, as opposed to anattack upon the organisms responsible for producing the slime.

The Enzyme We have discovered that slime accumulation can be controlledor reduced by the addition to industrial waters containing levan-formingorganisms by the addition to such waters of the enzyme levan hydrolase(sometimes called levan polyase).

The enzyme levan hydrolase is produced by a variety of microorganisms.It is possible that some of the enzyme is inherently produced in certainindustrial water systems. This may account, in part, for the lack ofcorrelation between total microbial count and slime formation. However,the conditions that exist in industrial waters such as white water donot ordinarily favor the production of substantial amounts of theenzyme. Consequently, it is necessary to isolate and grow an appropriateenzyme-producing microorganism under conditions that favor maximumproduction of the enzyme. Microorganisms that are reported to producethe enzyme levan hydrolase include the following:

Rhodotorula sp.

Azotobacter sp.

Bacillus sp.

Arthrobacter sp.

Micrococcus sp.

Pseudomonas sp. The form of the enzyme will vary depending upon theparticular source of the enzyme that is cultured. For example, levanhydrolase produced by Azotobacter sp. produces an extra-cellular enzymewhereas the yeast Rhodotorula sp. produces enzyme within or on the cellstructure.

Use of Levan Hydrolase to Control Slime Accumulation Levan hydrolase isused to control slime accumulation by the intentional addition of theenzyme to industrial waters containing slime-forming microorganisms. Theconcentration of the enzyme can vary widely and will depend upon suchfactors as the type of industrial water being treated (e.g., whitewater), the conditions of treatment (e.g., temperature and pH of thewater), the source of the enzyme (e.g., Rhodotorula sp.), and the enzymeactivity. When using the enzyme in the form of dead cells of the yeastRhodotorula sp. (at an activity of 20 units per gram), concentrations offrom about 1 ppm up to 100 ppm of the cells in water are effective.Above 500 ppm, there is sometimes a tendency for the enzyme to discolorthe water being treated. Good results are generally obtained atconcentrations of 05-20 ppm (e.g., 1-10 ppm). Such cells areconveniently used by drying the cells, diluting them with a salt (e.g.,a metal sulfate such as sodium sulfate) to a concentration of, forexample, ten percent by weight, and thereafter adding the resultingmixture to the water to be treated. if desired, the enzyme can be addedto the water alone or mixed with other water additives (e.g., biocides,biostats, buffers, and the like).

The present invention is further illustrated by the following specificexamples. Unless otherwise: indicated, all parts and percentages are byweight.

Experimental Preparation of the Enzyme Levan Hydrolase a. Maintenance ofRhodotorula sp.

A species of the yeast Rhodotorula was isolated using enrichment culturetechniques. This microorganism was found to be capable of decomposinglevan and was thereafter maintained in soil tubes and on an agar slantmedium having the following composition in grams per liter.

Beef Extract (Difco) 10.0

Yeast Extract (Difco) 3.0

Peptone (Difco) 10.0

Sucrose 50.0

Sodium Chloride 5.0

In making the slant medium, sufficient distilled water was added todissolve the above listed ingredients. The resulting solution wasadjusted to pH 7.5 with 2.5 M sodium hydroxide and l.2%/W/V agar (Difco)was added. The suspension was then made up to volume with distilledwater. The agar was melted by heating the medium and boiling as short atime as necessary. The medium was dispensed in test tubes and sterilizedat 121? C for 20 minutes. The tubes were cooled in a slanted position toobtain a long slant and short butt.

The slant medium was inoculated from soil tubes and the cultures ofRhodotorula sp. were incubated at 30 C. for from four to five daysbefore use.

b. Preparation and Inoculation of'Seed Medium Two ml of a 5 ml sterilewater suspension of a 4- to S-day-bld Rhodotorula culture was used toinoculate 50 ml of seed medium in a 250 ml baffled Erlenmeyer flask. Theseed medium contained the following ingredients in grams per liter ofaqueous medium:

Sucrose 20.0

Black S ap Molasses 15.0

Yeas Extract (Difco) 5.0

Peptone (Difco) 12.0 The pH of the medium was adjusted to 6.7.

The medium was sterilized by autoclaving at 121 C. for 20 minutes andthen cooled as rapidly as possible. After inoculation, the cultures wereincubated on a incubator-shaker (New Brunswick Gyrotory Model G-25)operating at about rpm and 27 C. for 24-26 hours.

c. Preparation and Inoculation of lnoculum Medium The contents ofone24-26 hour 50 ml culture of seed growth was used to inoculate 500 ml ofinoculum medium in a two-liter baffled Erlenmeyer flask. The inoculummedium contained the following ingredients in grams per liter of aqueousmedium:

Black Strap Molasses 100.0

Epsom Salts 0.6

Yeast 2.5

Corn Steep Liquor 20.0

Diammonium phosphate- 10.0

The solution was adjusted to pH 6.7, sterilized by autoclaving at 121 C.and cooled as rapidly as possible. After inoculation, cultures wereincubated for 22-24 hours as described for the seed medium.

d. Preparation and Inoculation of Production Medium The contents of two22-24 hour 500 ml cultures of inoculum growth was used to inoculate 8kg.of production medium in a 14 liter fermentor. The fermentor was fullybaffled and agitation was provided by means of two fixed vane,turbine-type, impellers 10 cm apart and 10 cm in diameter. Sterile airwas introduced through a one-fourth inch I.D. open pipe located directlybeneath the bottom impeller. The fermentor was operated at an agitatorspeed of 700 rpm with air flow at one volume per volume of medium perminute. The temperature was controlled at 27 C: 2 C. by means of acirculating water bath. Foam control was accomplished by the use ofanitfoam agents.

During the production phase, growth was monitored by measurement of pHand packed cell volumes. The latter were measured in calibrated conicalcentrifuge tubes. Levan hydrolase activity was measured by amodification of the tetrazolium chloride assay described by Avigad, et21].". Levan substrate for the enzyme assay was prepared usingAerobacter levanicum according to the method of Avigad but isolation ofthe levan was omitted. The form of levan obtained in the isolationprocedure is not decomposed appreciably by the enzyme produced by ourstrain of Rhodotorula. The enzyme assay procedure was as follows:

Rhodotorula cells were separated from fermentor broth by centrifugationor by ethanol precipitation. The cells were diluted in 0.05 Mmorpholinopropane sulfonic acid buffer (pH 6.5). One ml of dilutedenzyme was added to each of three test tubes. The tubes were placed in a40 C. water bath for 5 minutes. One ml of substrate containing at leastmicromoles of levan (determined as fructose) was added to the first twotubes, the third being used as a reagent blank. After exactly 30 minutesreaction time, 2.0 ml of 0.5 M sodium hydroxide were added to all threetubes. One ml of substrate was added to the reagent blank. Then 2.0 mlof 10 percent zinc sulfate heptahydrate were added to all tubes. Theprecipitate was removed by either filtration or centrifugation, and 1.0ml of the supernate was added to each of three test tubes. After adding1.0 ml of 1 1. Avigad, L., Ruth Zelikson, and S. Hestrin, SelectiveDetermination of Sugars Manifesting Enediol Isomerism by Means ofReaction with Tetrazolium. Biochem. J. 80, 57-61 (1961). 2. Avigad, L.,Methods in Carbohydrate Chemistry V, 161-165. Academic Press, New York(1965). percent 2,3,5-tripheny1-ZI-I-tetrazolium chloride, the tubeswere placed in a 40 C. water bath for five minutes. Then 1.0 ml of 0.5 MNaOH was added to each tube. After exactly minutes, 5 ml of a 10 percentby volume glacial acetic acid in methanol solution was added to stop thereaction. The optical density was read at 580 my. in aspectrophotometer.

Tubes which contain a red precipitate in the above assay must bediscarded and the cells diluted further for reassay. Units of activityper unit weight is defined as the product of dilution factor and opticaldensity at 580 millimicrons.

When assays indicated that the yield of levan hydrolase reached amaximum, the fermentation was terminated and the cells of Rhodotorulawere collected.

e. The Recovery of Levan Hydrolase A total weight of 45,300 grams ofbroth was collected from six production fermentors 44 hours afterinoculation. The broth was passed through a separator which separatedthe broth into a wort phase and a yeast concentrate. The wort phase wasdiluted to the original volume of broth and again passed through thecentrifuge. The yeast concentratesfrom the first and secondcentrifugations were combined. The second wort, which had a very lowyeast packed cell volume, was discarded. The total yeast concentrate wasdiluted to 2.5 times its volume with water containing one percent of anon-ionic surfactant (commercially available as DC- 161 from EconomicsLaboratory, Inc. of St. Paul, Minnesota). It has been found that theactivity and appearance of the final product is improved by thesurfactant wash. The suspension was again passed through the separatorand the wort discarded. The yeast concentrate was diluted with denaturedalcohol (95 percent ethanol) until the alcohol concentration of themixture reached 70 percent. A precipitate formed and was thereaftercollected by filtration. The filtrate was discarded and the cake driedin a vacuum oven at 40 C. The cake was then ground to a fine powder (80mesh). A total weight of 736 grams of yeast was collected having a levanhydrolase activity of units per gram.

Experimental Procedures for Examples 1-5 The apparatus used in theseexamples was similar to that described by Leckeys'" In this experimentalsystem,

paper mill white water (synthetic or natural) is passed over wood boardsin such a manner as to encourage the build-up of a mass of wood pulp,insoluble inorganic materials and microbial cells. The mass is heldtogether by bacterial slime so that it is similar in nature to thesubstance found to be a problem in paper mills.

Two boxes were used in these examples, one for testing the enzyme andone for control purposes. Each box was built to accommodate five gallonsof white paper and five slirne boards positioned just above the surfaceof the water at an angle of inclination of about 45. The white water wasrun over the boards from a horizontal manifold attached to a submersiblepump. Five holes in the manifold were located about 2 inches above thefive boards. The flow rate of the water from the manifold variedconsiderably depending upon slime accumulation, but averaged roughly 500ml per minute. In examples 1-3, white water was obtained from a papermill on a daily basis. The initial sample consisted of ten gallons tofill both boxes. Thereafter, one gallon 01 fresh white water was addedafter 3. Leckey, C. R., The Slime Board Method of Paper Mill ToxicantEvaluation, TAPPI, 43, 781-783 (1960). 1 gallon had been removed. Thelatter was used for determination of microbial plate count, pH, andqualitative analysis for fructose. The temperatures of the boxes wereallowed to reach their own levels to simulate the variable conditionsfound in paper mills. Average temperature values are given in theresults. On some days, temperatures varied over as much as a ten degreerange.

In order to allow for differences in box geometry and pumps, the boxreceiving the enzyme was alternated with each experiment. Enzyme wasadded according to apparent need.

Plate counts were made using the following medium at a pH of 6.7-7.0:

Plate Count Agar (Difco) 2.4 grams Brom Cresol Purple (alcoholicsolution 1.6%) 0.1 ml CaCl, H O (0.026% solution) 1.0 ml Mg SO, 7H,O(0.05% solution) 1.0 ml

Distilled water to ml The medium was sterilized at 121 C. for 30minutes.

All plates were incubated, after inoculation, at 45 C. for two days.Colonies were counted by means of a New Brunswick Colony Counter, ModelC-1 10.

Qualitative tests for fructose were made using the resorcinol-thioureareagent method described by Avigad In this test, both fructose as levanand free fructose are detected if present. Samples were also examinedfor the presence of free fructose, alone, using 2,3,5-triphenyl-2H-tetrazolium chloride reagent (TTC). The latter reagentcan also be used for detection of glucose, but the reaction can be mademore specific for ketose sugars by the use of a short reaction time.

2. Avigad, L., Methods in Carbohydrate Chemistry V,

Academic Press, New York 1965). A positive resorcinol test (levan plusfree fructose) and a negative TTC test (free fructose, only), means thatonly levan is present.

In Examples 4 and 5, synthetic white water was sterilized and theninoculated with a known levan-producing bacterium, Aerobacter levant'cum(ATCC No. 15552) to study the effect of Rhodotorula levan hydrolase cellconcentration on bacterial slime in a simulated paper mill system. Muchmore rapid experimental results can be obtained in this system than withnatural paper mill white water. The synthetic white water contained thefollowing ingredients in grams per liter:

Powdered cellulose 2.0 Bactopeptone 10.0 Sucrose 50.0 Starch 0.3 ChinaClay 0.3 Titanium dioxide 0.2 Animal glue 0.0l Wet strength resin 0.3Sodium aluminate 0.2 Rosin size 0.2

The pH of the synthetic white water was adjusted to pH 5.0 withtechnical grade sulfuric acid.

In Examples 4 and 5, each white water box was cleaned with 70 percentethanol before addition of 20 liters of the sterile synthetic whitewater. The white water was then inoculated with 2 liters of A. levanicumculture. The bacteria for the inoculum were grown in one liter baffledflasks containing 300 ml of synthetic white water at 27 C. for 18 hours.All other aspects of Examples 4 and 5 were carried out as described forexperiments with paper mill white water.

Thin layer chromatography on cellulose was used to establish that thefructan observed in paper mill white water was identical, at least inpart, to that formed by Aerobacter levanicum, and that it was decomposedin the same manner as the bacterial levan.

The results of Examples l-5 are set forth in the tables which follow.

Example 1 This example was run on White Water from a Fourdrinier PaperMachine of Paper Mill A.

Untreated White Water Days of Temp. Colonies Resorcinol- Run C. pH perml Thiourea TTC 3 50 7.9 2.3)(10 4 42 8.4 6.6Xl" 5 46 8.5 7.9 l0

6 41 8.4 l.9 l0' 7 44 8.3 8.9 l0' IO 49 8.4 1.2Xl0 ll 49 8.5 9.6)(10 1249 8.5 7.9Xl0 I4 49 8.3 l.2) l0 I7 49 8.6 9.3Xl0

Enzyme Treated White Water Days of Temp. Colonies Resorcinol- Run C. pHper ml Thiourea TTC 3 45 7.4 2.2 l0 not done 4' 36 8.4 7.0Xl0 Do. 5' 428.1 3.9Xl0 Do. 6* 42 7.9 4.4Xl0 Do. 7 34 7.6 9.3Xl0 Do I0 48 7.6 9.8 l0D0 ll 48 7.6 5.4 (l0 D0. 12 44 7.8 5.5Xl0' D0. 14 48 7.8 l.2XlO" D0. 1749 7.0 4.3Xl0' Do.

' 2250 ppm enzyme added with levan hydrolase activity of 2 units/- gram.Enzyme additions varied according to apparent effect with trend toreduced amounts. In all cases indicated amount represents average oftotal addition.

Example 2 This Example was run on White Water from a Cylinder PaperMachine of Paper Mill A.

Untreated White Water Days of Temp. Colonies Resorcinol- Run C. pH perml Thiourea TTC 1 47 4.3 3.3)(10 3 46 6.2 3.3Xl0 4 48 6.9 7.9Xl0 7 444.6 4.0Xl0' 8 46 5.6 2.3Xl0' 9 48 4.5 3.8 l0 x l l 50 4.3 4.7 l0" 16 494.3 6.5)(10 Enzyme Treated White Water Days of Temp. ColoniesResorcinol- Run "C. pH per ml Thiourea TTC 1 42 4.3 No count No tests 346 5.8 3.7 l0 4 44 6.9 3.6Xl0 7 45 4.8 2.8Xl0' 8 46 5.3 3.5 l0" 9" 49 4.3.8Xl0" 10* No samples taken 11 50 7.5 5.9Xl0 l3 No samples taken [6 497.8 4.l l0

2250 ppm enzyme added with levan hydrolase activity of 2 units/- gram.

Example 3 This Example was run on White Water from a Fourdrinier PaperMachine of Paper Mill B.

Untreated White Water Days of Temp. Colonies Resorcinol- Run pH per mlThiourea TTC 1 28 7.8 1.3Xl0 2 44 8.6 4.9)(10' 6 50 8.4 3.8Xl0 7 46 8.42.0)(10' 8 44 8.5 4.lXl0 9 49 8.5 2.1Xl0" I0 46 8.5 3.9Xl0 l3 4] 8.3 l.8(l0 I4 43 8.3 2.4 l() 30 15 42 8.2 2.6 l0

Enzyme Treated White Water Days of Temp. Colonies Resorcinol- Run C. pHper ml Thiourea TTC 1' 24 7.8 l.4XlO 2 42 7.9 5.7Xl0 6 45 8.3 4.0Xl0" 746 8.1 3.8X 10 8* 44 8.2 1.4X ]0 9* 46 8.3 3.2Xl0 10 4l 8.0 3.lXlO 13*36 8.7 2.8Xl0 14 39 8.5 2.3Xl0" 15 39 8.4 2.9Xl0 300 ppm enzyme addedwith levan hydrolase activity of2 units/gram Example 4 This Example wasrun on Synthetic White Water Inoculated with Aerobacter levanicum (ATCC15552).

Untreated White Water Days of Temp. Colonies Resorcinol- Run C. pH perml Thiourea TTC 3 45 5.8 2.8Xl0" 4 42 6.9 2.2Xl0" 5 42 7.3 2.0Xl0 6 437.2 2.8Xl0 7 4l 7.6 2.8Xl0 l0 7.8 2.8Xl0 l l 45 7.5 3.]Xl0 I2 47 7.63.0Xl0

Enzyme Treated White Water Days of Temp. Colonies Resorcinol- Run C. pHper ml Thiourea TTC 3' 45 6.3 2.3 l0 4 44 7.] 2.9Xl0" 5* 42 7.5 2.9 l0 644 7.4 3.7Xl0 7 40 7.8 2.0Xl0" 10* 7.7 2.7 l0 l l 37 7.9 3.0 l0 12' 467.3 2.8 |0" l5 ppm enzyme added with levan hydrolase activity of 2units/gram Example 5 This Example was run on Synthetic White WaterInoculated with Aerobacter levanicum (ATCC 15552).

Untreated White Water Days of Temp. Colonies Resorcinol- Run C. pH perml Thiourea 'l'l'C 3 4| 6.3 23x10 4 42 7.1 2.3Xl 5 44 7.8 3.3x 6 44 8.42.8X10 7 48 8.2 2.0Xl0 8 48 Not done 9 46 Not done Enzyme Treated WhiteWater Days of Temp. Colonies Resorcinol- Run C. pH per ml Thiourea TTC 341 5.9 2.8Xl0" 4* 42 6.8 2.l 10 5 44 7.4 2.2Xl0 6 42 7.9 2.8)(10 7' 448.0 2.7 l0 8* 44 Not done 9* 44 Not done t 5 ppm enzyme added with levanhydrolase activity of units/gram.

The beneficial results which are obtained can be understood by referenceto tests run on white water of Paper Mill B (e.g., see Example 3). Slimeremoval from the boards and prevention of slime build-up was observed inthe enzyme treated system as contrasted to the untreated system. Inaddition, a small difference in temperature between treated anduntreated water was noticed with the lower temperature being obtained inthe treated water. Considerable foam formation consistenly occurred inuntreated water but was absent in the enzyme treated water. Similarresults were obtained with the white water of Paper Mill A.

What is claimed is:

1. A composition for treating slime in industrial waters with the enzymelevan hydrolase, said composition consisting essentially of:

a. dried, dead cells of the yeast Rhodotorula sp., said cells havinglevan hydrolase activity when mixed with unisolated levan and testedaccording to the tetrazolium chloride assay;

b. sodium sulfate, admixed with said dried, dead cells as a diluenttherefor.

2. A composition according to claim 1 wherein said composition furthercontains an additive selected from the group consisting of a biocide, abiostat and a buffer.

3. A composition according to claim 1 wherein said.

dried dead cells comprise 10 percent by weight of said composition, thebalance to percent consisting of sodium sulfate.

UNITED SIAT ES PATENT OFFICE CERTIFICATE OF CORRECTION en No, 3,773,623Dated November 20, 1973 Inven tor(s) Herbert J. Hatcher'et a1 It iscertified that error appears in the above-idntified patent and that saidLetters Patent are hereby corrected as shown below:

- In columril, line 62, should appear as a superscript In colum1 1 4,line 64, should appear as asuperscript In columxg 5, line 68, shouldappear as a superscript;

In column 6, line 9, "pape should read waterl--..

'In column 5, lines'l8 23; column 6, lines-22 ,24; and.

colunm 6, lines 58 60, through (1965) '.","the text "of footnotes 91","2", and "3", should have been placed at the bottom of columns '5 and 6in the order of their appearancein the text of these columns. v

In colum x 6, line 51, should appear as a superscript In the tableheaded "Untreated White Water" in."Example 2",- column 7, line 62 et sethe headings for the various columns ofgthe table should notbridge'columns? and-8'.

Thus, the heading for. the first column of the table should read --Daysof Run--, for the second column 7 I "Temp. -C for the fourth,column,'--Colonies per ml-- and for the fifth column,--Resorcindl-Thiourea---.

In column 8, line 33, "3 0"-shouldbe a plus sign Signed and sealed this119611 day of r-ey 19m;-

SEAL.) Attest:

EDWARD I LFLETCIBEILJR. I Q G. MARSHALL DANN Attesting OfficerCommissioner of Patents-

2. A composition according to claim 1 wherein said composition furthercontains an additive selected from the group consisting of a biocide, abiostat and a buffer.
 3. A composition according to claim 1 wherein saiddried dead cells comprise 10 percent by weight of said composition, thebalance to 100 percent consisting of sodium sulfate.